Acute myeloid leukemia (AML) is a heterogeneous disease with diverse gene mutations and chromosomal abnormalities. Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for approximately 24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia. The encoded proteins, RUNX1 and CBFbeta, form a heterodimer to regulate gene expression, and they are both required for hematopoiesis in vertebrate animals from zebrafish to man. Extensive clinical studies have demonstrated that CBFB-MYH11 and RUNX1-ETO, the two common fusion genes in CBF leukemia, are the best biomarkers for diagnosis, prognosis, and residual disease monitoring of CBF leukemia patients. Even though CBF leukemias have better initial remission rate and better prognosis than most AML cases, current chemotherapy is associated with significant morbidity and mortality, and the long-term survival (>5 year) is only around 50-60%. Over the years we have used mouse models and a variety of research tools to characterize the CBFB-MYH11 fusion gene, determine the effect of the encoded protein, CBFbeta-SMMHC, on normal hematopoiesis, and understand the leukemogenesis process associated with the fusion gene. We have generated both conventional and conditional knock-in mouse models to study CBFB-MYH11. Using these mouse models we have demonstrated that Cbfb-MYH11 dominantly inhibits Runx1 and Cbfb function during definitive hematopoiesis, resulting in complete lack of definitive hematopoiesis in the heterozygous Cbfb-MYH11 knockin embryos. We also showed that Cbfb-MYH11 is necessary but not sufficient for leukemia, and we were able to identify cooperating genetic events in the mouse models. We have generated knock-in mouse models expressing truncated Cbfb-MYH11 to determine the importance of functional domains of CBFbeta-SMMHC. Overall our lab has been recognized in the field as the major contributor to the understanding of CBFB-MYH11 leukemia. In the last fiscal year we focused on three specific aims in this project. In the first aim we generate mouse models to study the mechanism of leukemogenesis by CBFB-MYH11. We were particularly interested in the contributions of the C-terminal domains of the CBFbeta-SMMHC fusion protein to leukemogenesis. The C-terminus of the fusion protein CBF&#946;-SMMHC contains domains for multimerization and transcriptional repression, both of which have been proposed to be important for leukemogenesis by CBF&#946;-SMMHC from in vitro studies. To test the role of the fusion proteins C-terminus in vivo, we generated knock-in mice expressing a C-terminally truncated CBF&#946;-SMMHC (CBF&#946;-SMMHC&#916;C95). Embryos with a single copy of CBF&#946;-SMMHC&#916;C95 were viable and showed no defects in hematopoiesis, while embryos homozygous for the CBF&#946;-SMMHC&#916;C95 allele had hematopoietic defects and died in mid-gestation, similar to embryos with a single-copy of the full-length CBF&#946;-SMMHC. Importantly, unlike mice expressing full-length CBF&#946;-SMMHC, none of the mice expressing CBF&#946;-SMMHC&#916;C95 developed leukemia, even after treatment with ENU. Our data indicate that the CBF&#946;-SMMHCs C-terminus is essential to induce embryonic hematopoietic defects and leukemogenesis. This result suggests that the C-terminal region can be considered for future development of targeted therapy for this type of leukemia. In the second aim we continued our development and characterization of novel compounds for targeted therapy against CBF leukemias. Our strategy is based on the hypothesis that the RUNX1-CBF&#946; interaction is critically important for CBF leukemia, and inhibition of this interaction can be therapeutically beneficial to the CBF leukemia patients. The novel compounds that we are developing that target RUNX1-CBF&#946; interaction will greatly enhance our ability to treat CBF leukemia patients with greater efficacy and fewer side effects than conventional chemotherapies, if we achieve our goals. In addition to CBF leukemia, recent collaborations with several groups indicate that we can broaden the potential applications of these compounds to other leukemia, and even some solid tumors. In the previous fiscal year (FY12) we identified benzodiazepine compounds that bind to both RUNX1 and CBF&#946;, and inhibit their functions. The finding that this class of compounds binds to RUNX1 and CBF&#946; was completely unexpected. The first goal of the aim was to identify the surfaces and residues of RUNX1 and CBF&#946; that interact with Ro5-3335 and understand how the compound inhibits RUNX1 and CBF&#946;. To do this, we have been taking a multi-pronged approach through collaborations with structural biologists and medicinal chemists. We have also been testing analogs of our lead compound, Ro5-3335, to identify more potent inhibitors. A bulk of our work in this second aim is in collaboration with the Rare and Neglected Diseases (TRND) program of NCATS, and we will hand off the project to TRND and clinical collaborators once the pre-clinical development phase is completed. The final aim of this project is to characterize relapsed CBF leukemia through sequencing. Relapse occurs in 30-70% of the patients with acute myeloid leukemia, and is a major reason for patient mortality. This is especially true for CBF leukemia, since the initial remission rate is very high. Treatment options for relapsed leukemia are limited, and the only potentially curative therapy for relapsed leukemia is hematopoietic stem cell transplantation, which is not available for every patient. It has been shown recently that mutations or genomic rearrangements taking place in a subset of leukemia cells can lead to relapse. If we can identify such mutations in CBF leukemias through genomic sequencing studies, we will be able to better understand the molecular mechanisms underlying relapse and design more effective treatments for such patients. Through collaborators in Ohio State University and MD Anderson Cancer Center, we have obtained DNA samples from multiple CBF leukemia patients and will conduct the sequencing experiments soon.